Clonal Immunoglobulin Heavy Chain Research Articles

Evaluation of next-generation sequencing versus next-generation flow cytometry for minimal-residual-disease detection in Chinese patients with multiple myeloma

PurposeTo evaluate the efficacy of next-generation sequencing (NGS) in minimal-residual-disease (MRD) monitoring in Chinese patients with multiple myeloma (MM).MethodsThis study analyzed 60 Chinese MM patients. During MRD monitoring in these patients’ post-therapy, clonal immunoglobulin heavy chain (IGH) rearrangements were detected via NGS using LymphoTrack assays. MRD monitoring was performed using NGS or next-generation flow cytometry (NGF), and the results were compared. Additionally, the sensitivity and reproducibility of the NGS method were assessed.ResultsThe MRD detection range of the NGS method was 10–6–10–1, which suggested good linearity, with a Pearson correlation coefficient of 0.985 and a limit of detection of 10–6. Intra- and inter-assay reproducibility analyses showed that NGS exhibited 100% reproducibility with low variability in clonal cells. At diagnosis, unique clones were found in 42 patients (70.0%) with clonal IGH rearrangements, which were used as clonality markers for MRD monitoring post-therapy. Comparison of NGS and NGF for MRD monitoring showed 79.1% concordance. No samples that tested MRD-positive via NGF were found negative via NGS, indicating the higher sensitivity of NGS. MRD could be detected using NGS in 6 of 7 samples before autologous hematopoietic stem-cell transplantation, and 5 of them tested negative post-transplantation. In contrast, the NGF method could detect MRD in only 1 sample pre-transplantation.ConclusionCompared with NGF, NGS exhibits higher sensitivity and reproducibility in MRD detection and can be an effective strategy for MRD monitoring in Chinese MM patients.

The Effect of Preemptive Rituximab Treatment in Patients with EBV Viremia after Solid Organ Transplantation on the Development of Post-Transplant Lymphoproliferative Disorder

The association between Epstein-Barr Virus (EBV) reactivation after solid organ transplantation (SOT) and Post-Transplant Lymphoproliferative Disorder (PTLD) is well described. There are currently no guidelines regarding the use of rituximab preemptively in patients with EBV viremia after SOT to reduce the risk of PTLD. We sought to determine whether rituximab in SOT patients with EBV viremia has benefit in preventing PTLD. This retrospective analysis of electronic medical record data identified SOT recipients who experienced EBV viremia post-transplant between January 2010 and February 2023 at Columbia University Irving Medical Center (CUIMC). Data queries were clinically reviewed. Logistic regression analysis was performed to investigate the relationship between predictor variables including rituximab administration, log-transformed EBV peak titer, duration of EBV viremia, and time from SOT to EBV reactivation, with the binary primary outcome of PTLD diagnosis status. The analysis encompassed both univariate and multivariate logistic regression. There were 2,168 patients who met the inclusion criteria of having EBV reactivation after SOT, out of 6,395 total SOT performed at CUIMC during this timeframe. Patient characteristics are described in table 1. The median peak EBV titer was 154 (range: not quantifiable - 5,335,816), the median duration of EBV viremia was 78 days (range: 1 - 6,980), and the median time from SOT to EBV reactivation was 688 days (range: 0-11103). Of the 2,168 patients, 185/2168 (8.5%) received rituximab for indications other than PTLD including EBV viremia, treatment of rejection, antibody desensitization therapy/immunosuppression peri-transplant, and other non-transplant related indications (e.g. lupus nephritis). Of the patients who received rituximab, 182/185 (98.4%) did not develop PTLD. The 3/185 (1.6%) patients who did develop PTLD were all ≤ 20 years-old at time of diagnosis, and had EBER(+), monomorphic PTLD, diffuse large B-cell lymphoma type, with a non-germinal center phenotype, involving the gastrointestinal tract. Two were CD20 positive, all 3 had partial weak CD30 positivity, and all 3 had clonal immunoglobulin heavy chain rearrangements. Among the patients who did not receive preemptive rituximab post-SOT, 195/1983 (9.8%) developed PTLD, whereas 1788/1983 (90.2%) did not. Prior rituximab therapy for any indication in patients with EBV viremia post-SOT was associated with lower risk of subsequent PTLD by univariate analysis (0.14% vs 8.39%, OR 0.15, p = 0.0013, 95% CI 0.048-0.48). Receipt of rituximab (OR 0.12, p=0.0002), log-transformed peak EBV titer (OR 1.10, p<0.0001), and duration of EBV viremia in months (OR 1.01, p<0.0001) were all independently associated with risk for PTLD by multivariate analysis (figure 1). The relationship between time from SOT to EBV viremia was not significantly associated with development of PTLD. This retrospective analysis suggests that administering preemptive rituximab in SOT patients with EBV viremia may lower the risk of developing PTLD. The fact that peak EBV titer and duration of EBV viremia remain associated with PTLD in multivariate analyses suggests that the benefit of rituximab may be independent of the degree or duration of EBV viremia. A prospective analysis of factors such as area under the curve of EBV exposure, HLA status, T cell clonotype diversity, organ type, and immunosuppression type, is needed to better understand individual risks for improved selection of those who would benefit from preemptive rituximab.

B-355 Measurable Residual Disease Detection in Mantle Cell Lymphoma (MCL) Subjects Using NGS B-Cell MRD Assay

Abstract Background Mantle Cell Lymphoma (MCL) is an aggressive, rare form of non-Hodgkin B-cell lymphoma. Measurable Residual Disease (MRD) assessment can provide useful information when assessing and tracking response to therapy, refining treatment and predicting clinical outcome for subjects with B-cell lymphoproliferative diseases. One method of MRD detection is to identify and track specific clonal immunoglobulin heavy chain (IGH) gene rearrangement sequences on a next-generation sequencing (NGS) platform. Here, we present the NGS-based B-cell MRD Assay with 10−6 sensitivity for detecting MRD in malignancies in mantle cell lymphoma (MCL). Methods Residual DNA samples from different specimen types (44 BM and 77 PB) at different post-treatment time points were obtained from anonymized 47 MCL subjects that had been enrolled in a study approved by the Spanish Group of Lymphoma and Autologous Stem Cell Transplantation (clinical trial: NCT02682641, publication reference: 10.1200/JCO.21.02321). 121 of these follow up samples were tested by the B-cell MRD Assay by tracking the clonal sequences detected in corresponding baseline samples. Among them, 90 follow up samples were paired PB and BM samples. The B-cell MRD Assay workflow consists of DNA extraction and PCR based library preparation with proprietary multiplex master mixes (MMx) targeting either IGH Framework 1 (FR1) or 3 (FR3) regions, which both track the patient- and tumor-specific CDR3 sequence. Purified libraries are then equimolar pooled and sequenced on Illumina’s NextSeq Dx platform. The FASTQ output files from sequencing are analyzed using Invivoscribe’s B-Cell MRD Software-NextSeqDx. Results from Invivoscribe were compared to the results from Salamanca lab that were generated by qPCR. Results Based on the results from B-cell MRD Assay, 115/121 (95%) samples were valid, 42/115 (37%) valid samples had, “MRD Detected”, and 73/115 (63%) samples had, “MRD Not Detected”, with an “MRD Detection” rate of 28% in PB and 50% in BM specimen types. When comparing MRD detectable status between paired PB and BM samples from 36 MCL subjects, 30/44 (68%) paired samples had same MRD call and were concordant, whereas 14/44 (32%) calls were discordant. More specifically, 12/14 (86%) discordant samples had PB specimen type with a “MRD Not detected” call, whereas the paired BM specimen showed a “MRD Detected” call. Overall, in comparison with the MRD results (n = 84) provided by Salamanca lab using qPCR method, the B-cell MRD Assay demonstrated concordance of 80%, PPA of 79%, and NPA of 80% for MRD detection in MCL samples. Conclusion Good concordance was observed between the qPCR used at Salamanca and Invivoscribe’s B-cell MRD assay. It was important to note that the BM specimen type was determined to be more sensitive in detecting MRD when compared to PB in assessment of paired PB and BM samples. These results demonstrate that the B-cell MRD Assay can be utilized for MRD detection in follow-up sample types for MCL subjects.

HIGH RATE OF METABOLIC COMPLETE RESPONSE AFTER LOW DOSE RADIOTHERAPY AND OBINUTUZUMAB IN EARLY STAGE FOLLICULAR LYMPHOMA: INITIAL RESULTS OF THE GAZAI STUDY (GLA 2018‐3)

Introduction: The FORT trial demonstrated a higher complete remission (CR) rate using 12 × 2 Gy compared to 2 × 2 Gy in Follicular Lymphoma (FL) (67% vs. 47%). This benefit resulted also in an improved progression free survival (PFS). The TROG 99.03 trial and the MIR study showed an improved PFS with the addition of Rituximab to radiation therapy of 30–40 Gy in early stage FL. Preclinical data suggest that the anti-CD20 antibody may act synergistic with radiation therapy and therefore enhance the radiation effect of low dose radiotherapy. The prospective, multicentric phase II GAZAI study of the German Lymphoma Alliance (GLA) investigated the efficacy of low dose radiation therapy (2 × 2 Gy) in combination with the anti-CD20 antibody Obinutuzumab in early stage nodal FL. The primary endpoint was the metabolic complete remission at the end of therapy. Methods: Patients with early stage FL grade 1/2 were recruited in 11 German centers. Ann Arbor stage I or II was confirmed by FDG-PET/CT. Patients received 1000 mg Obinutuzumab flat dose intravenously in weeks 1, 2, 3, 4, 8, 12 and 16. An interim CT scan was performed in week 7 for response evaluation and subsequent treatment planning. Radiation therapy was applied to the initial involved sites (site of diagnostic surgical intervention and PET positive sites) in week 9. The radiation dose was 2 x 2 Gy on two succeeding days. A FDG-PET/CT was performed at the end of therapy in week 18 for evaluation of the metabolic and morphologic response. Minimal detectable/residual disease (MRD) in peripheral blood was monitored at baseline and at week 18 by allel-specific RQ PCR targeting t(14;18) translocations and clonal immunoglobulin heavy chain (IGH) rearrangements. Results: Of 89 patients included in the study, 54 entered the treatment phase showing FDG-PET-positive lymph nodes qualifying for the primary endpoint. At week 18, metabolic CR (Deauville score (DS) <3) was seen in 46/53 patients (87%; one patient only had a CT without FDG-PET). Partial metabolic remission with a DS 3 was seen in 3 patients (6%) and 3 patients showed a DS 4. Morphologic (CT based) CR/CRu according to Cheson 1999 criteria was seen in 21/54 patients (39%) at week 7 and in 49/54 patients (91%) at week 18. Morphologic PR was seen in 17 patients (32%) at week 7 and in 4 patients (7%) at week 18; one patient (2%) showed progressive disease (metabolic and morphologic) outside of the radiation field. 13/54 patients (24%) were initially MRD positive. All but one patient converted to MRD negativity at week 18. Conclusions: Low dose radiation therapy using 2 × 2 Gy in combination with Obinutuzumab is highly effective in early stage FL. Longer follow-up is necessary to investigate if this effectivity also results in a prolonged PFS. Currently, the FORTplus trial is looking for non-inferiority of this regimen in comparison to a conventional radiation dose (12 × 2 Gy) combined with Rituximab. The research was funded by: Roche Pharma Keywords: combination therapies, indolent non-Hodgkin lymphoma, radiation therapy Conflicts of interests pertinent to the abstract K. Herfarth Research funding: Roche Pharma Educational grants: Roche Pharma C. W. Scholz Consultant or advisory role: Roche Pharma Honoraria: Speakers Honoraria Roche Pharma Educational grants: Roche Pharma K. Hübel Consultant or advisory role: Scientific advisory board Roche Pharma Honoraria: Speaker Honoraria Roche Pharma Research funding: Roche Pharma C. Buske Consultant or advisory role: Roche Pharma Honoraria: Roche Pharma Research funding: Roche Pharma J. Dürig Consultant or advisory role: Scientific advisory board Roche Pharma Honoraria: Speakers Honoraria Roche Pharma Educational grants: Roche Pharma G. Lenz Consultant or advisory role: Advisory board Roche Pharma Honoraria: Speakers honoraria Roche Pharma Research funding: Roche Pharma C. Pott Research funding: Roche Pharma M. Dreyling Consultant or advisory role: Roche Pharma Honoraria: Speakers Honoraria Roche Pharma Research funding: Roche Pharma

Disseminated intramuscular anaplastic plasma cell tumour in a dog

AbstractA 12‐year‐old, neutered, male Jack Russell terrier was diagnosed with a disseminated intramuscular anaplastic round cell tumour with primary hepatic, splenic, subcutaneous and medullary involvement. The tumour was confirmed by histopathology, immunohistochemistry and polymerase chain reaction analysis of antigen receptor gene rearrangements to be of plasma cell origin. Tumour cells were lambda light chain‐positive, multiple myeloma 1‐positive and pan‐B marker‐negative (paired box protein 5, CD20 and CD79) and showed clonal immunoglobulin heavy chain gene rearrangement. Computed tomography did not identify osteolytic lesions. Plasma cells represented 14% of bone marrow cells. Serum and urine electrophoresis were not performed because of a normal serum globulin concentration. To the authors’ knowledge, this is the first case report describing a disseminated diffuse intramuscular anaplastic plasma cell tumour in a dog without evidence of multiple myeloma on initial presentation or progression to multiple myeloma or plasma cell leukaemia during follow‐up.

Combining MYD88 L265P mutation detection and clonality determination on CSF cellular and cell-free DNA improves diagnosis of primary CNS lymphoma.

Diagnosis of primary central nervous system lymphoma (PCNSL) is challenging, and although brain biopsy remains the gold standard, cerebrospinal fluid (CSF) constitutes a less invasive source of lymphomatous biomarkers. In a retrospective cohort of 54 PCNSL cases tested at diagnosis or relapse, we evaluated the contribution of immunoglobulin heavy chain (IGH) gene clonality and MYD88 L265P detection on both CSF cell pellets and supernatants, in comparison with cytology, flow cytometry, interleukin (IL)-10 and IL-6 quantification. Clonality assessment included a new assay to detect partial IGH-DJ rearrangements. Clonal IGH rearrangements and/or MYD88 L265P mutation were detected in 27 (50%) cell pellets and 24 (44%) supernatant cell-free (cf) DNA. Combining analyses on both compartments, 36 (66%) cases had at least one detectable molecular marker, present only in cfDNA for 9 (16%) of them. While cytology and flow cytometry were positive in only 7 (13.0%) and 9 (17.3%) cases respectively, high IL-10 levels were observed in 36 (66.7%) cases. Overall, taking into account molecular and cytokine results, 46/54 (85%) cases had at least one lymphomatous biomarker detectable in the CSF. These results show that this combination of biomarkers evaluated on both cell pellet and supernatant CSF fractions improves significantly the biological diagnosis of PCNSL.

Clinical Significance of IGH Clonality Analysis with RNA Sequencing in B-Cell Precursor Acute Lymphoblastic Leukemia

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common pediatric malignancy. Using minimal residual disease (MRD) and genetic subtypes, risk stratification algorithms have considerably improved clinical outcomes. However, despite being in a better prognostic group, a non-negligible number of relapses occur, and prognostic markers for these patients are required for more effective treatment strategies. RNA sequencing (RNA-seq) is a useful tool for detecting patient-specific immunoglobulin heavy chain (IGH) junctional sequences (Li et al., Leukemia. 2020), which can be informative for evaluating the nature of leukemic clones. Here, we analyzed IGH clonality of pediatric BCP-ALL using RNA-seq data and revealed its validity for evaluating disease characteristics and impact on prognosis. Methods: We analyzed RNA-seq data of 136 pediatric BCP-ALL samples at diagnosis and 46 samples at relapse from the TARGET ALL Expansion Phase 2 cohort. Using MiXCR (Bolotin et al., Nat Methods 2015) and Vidjil (Duez et al., PLoS One 2016), we detected IGH complete VDJ and incomplete DJ rearrangements. Then, we estimated the expected number of IGH rearrangements corresponding to each read count (E-value) by power-law model. Finally, we identified leukemic "disease clones” with E-values less than 0.01 (Fig. 1a). Results: First, we detected a total of 55,518 IGH clones, of which 48,555 were complete rearrangements and 6,963 were incomplete. In total, 351 disease clones (median 2, range 0-8 per sample) were identified by power-law model: 193 clones were complete rearrangements, and 158 clones were incomplete. Interestingly, the numbers and compositions (complete or incomplete rearrangements) of disease clones varied significantly between samples. Thus, we defined three clonality patterns and classified each sample based on identified disease clones: "Undetectable disease clones (UDC)” if the sample had no disease clone (n=18); "Incomplete Disease Clones (IDC)” if there were one or more disease clones with incomplete rearrangements (n=72); and "Complete Disease Clones (CDC)” if all disease clones of that sample have complete rearrangements (n=46). Gene Set Enrichment Analysis (GSEA) indicated a strong enrichment for hematopoietic stem cell (HSC) and multipotent progenitor (MPP) signatures in UDC and pro-B cell signatures in IDC. Similarly, CIBERSORTx (Newman et al., Nat Biotechnol 2019) showed higher proportions of HSCs, MPPs, and pre-pro B cells in UDC and pro-B cells in IDC. Furthermore, there was a significant enrichment in pathways promoting cell cycle progression in IDC. Of note, in high hyperdiploid (HHD) cases, which are typically associated with a favorable prognosis, event-free survival (EFS) was significantly lower in UDC and IDC (p=0.01) (Fig. 1b). Moreover, even in MRD-negative HHD patients, those with UDC and IDC had significantly worse EFS (p=0.01) and overall survival (p=0.03) (Fig. 1b). Finally, we compared the primary and relapse clones in 46 patients. As a result, 37 patients had one or more disease clones at diagnosis, and 29 (78%) had these primary disease clones at relapse. On the other hand, 9 (24%) patients showed relapse-specific disease clones, which should lead to relapse in these cases. The other 9 primary patients with UDC had no disease clone also at relapse. Discussions: We identified three clonality patterns, UDC, IDC, and CDC based on RNA-seq analysis of the IGH rearrangement status. According to the findings of GSEA and CIBERSORTx, leukemic cells of UDC, IDC, and CDC patients have nature of progenitors to pre-proB cells, pro-B cells, and more mature cells, respectively. These findings are consistent with the knowledge that IGH rearrangement occurs during pro-B to pre-B maturation in normal B-cells. Patients with immature clonality status, UDC and IDC, were associated with poor prognosis in high hyperdiploid subtype and could be a potential prognostic marker in MRD-negative patients. In addition, we revealed that primary disease clones contributed to relapse in 78% of cases, while relapse-specific clones were implicated in relapse in 24% of cases. Additionally, UDC clonality pattern was carried over at relapse. These findings shed light on the mechanisms of leukemic clonal evolution and relapse, and suggest that the status of IGH rearrangement may serve as a novel prognostic marker in BCP-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intraepithelial cytotoxic lymphocytes are associated with a poor prognosis in feline intestinal T-cell lymphoma.

The expression of cytotoxic molecules in feline intestinal T-cell lymphoma cells was examined immunohistochemically using endoscopic samples of 50 cases. Cases included 14 large-cell lymphomas (LCLs) and 36 small-cell lymphomas (SCLs). Most LCL and some SCL exhibited marked erosion and villous atrophy. Clonal T-cell receptor (TCR) gene rearrangement was detected in 10/14 (71%) LCL cases and 33/36 (92%) SCL cases. No clonal immunoglobulin heavy chain (IgH) gene rearrangement was detected. Immunohistochemically, all cases were positive for CD3 and negative for CD79α, CD30, CD56, and Foxp3. LCLs were positive for CD8 in 13/14 cases (93%), T-cell intracellular antigen 1 (TIA1) in 14/14 cases (100%), and granzyme B in 6/14 cases (43%). SCLs were positive for CD8 in 28/36 cases (78%), TIA1 in 33/36 cases (92%), and granzyme B in 2/36 cases (6%). TIA1- and granzyme B-positive neoplastic lymphocytes were predominantly observed in the mucosal epithelium of 10/50 cases (20%) and 6/50 cases (12%), respectively. No significant differences in survival time were found based on cell size or epitheliotropism. However, cases with TIA1+ and/or granzyme B+ neoplastic lymphocytes predominantly in the mucosal epithelium had significantly shorter survival times (P < .05), suggesting that mucosal epithelium infiltration of neoplastic cells with a cytotoxic immunophenotype is a negative prognostic factor. Therefore, intraepithelial cytotoxic lymphocytes may be associated with mucosal injury and impaired intestinal function, leading to a poor prognosis in cats with intestinal T-cell lymphoma.

P1245: CLINICAL IMPACT OF IMMUNOGLOBULIN HEAVY CHAIN REPERTOIRE IN MANTLE CELL LYMPHOMA: A STUDY FROM THE FONDAZIONE ITALIANA LINFOMI (FIL) PHASE III MCL0208 TRIAL.

Background: Mantle cell lymphoma (MCL) is characterized by a highly restricted immunoglobulin heavy chain (IGH) gene repertoire with stereotyped IGHV CDR3s, strongly implying a role for antigen-driven selection. Nonetheless, differently from chronic lymphocytic leukemia, where the prognostic role of IGH parameters is known since decades, published data are less consistent in MCL. Recently, LRPAP1 autoantibodies were detected in 13% of patients from the European MCL Network trials and were associated with superior outcome, while the autoreactivity of IGHV 4-34 antibodies is largely known. Aims: We aimed at characterizing the IGH VDJ repertoire and its putative clinical relevance in a large, prospective, trial of the Fondazione Italiana Linfomi (FIL) enrolling younger MCL patients. Methods: IGH clonal rearrangements detection was performed by Sanger sequencing on bone marrow or peripheral blood samples collected for minimal residual disease purposes in FIL MCL0208 trial (NCT02354313). This is a prospective, randomized phase III trial comparing lenalidomide maintenance vs observation after an intensive cytarabine containing chemo-immunotherapy followed by autologous transplantation in frontline <66 year-old MCL patients. IMGT/V-QUEST tool was used to annotate IGHV-D-J and mutational status (cut-off 98%) according to ERIC guidelines. Finally, these biological data were integrated with published clinical, pathological, and mutational data from the trial (current median follow-up 38 months). Results: A clonal IGH productive rearrangement was detected in 209/300 patients (70%), resulting in 159 (76%) unmutated (UM) and in 50 (24%) mutated (M) cases, as expected. Overall, VDJ usage included 29 IGHV families, with IGHV 3-21 (#45, 22%), 4-34 (#33, 16%), 3-7 (#15, 7%), 3-23 and 5-51 (#12 each, 6%), 4-39 and 4-59 (#10 each, 5%) as the most frequent groups, together representing the 66% of the analyzed series (Figure 1A). Interestingly, patients carrying IGHV 3-21 were often characterized by low Ki-67 index (Ki-67 <30%: 86% vs 64% of other cases, p<0.01) and low risk MIPI score (73% vs 43%, p=0.001), while no case of blastoid morphology was registered among the IGHV 4-34 patients (vs 89%, p<0.05). Moreover, by grouping together these two rearrangements, statistically significant associations were found with several biomarkers of favorable prognosis (low Ki-67, p<0.01; non-blastoid, p<0.05; absence of TP53 aberrations, p<0.05, or KMT2D mutations, p<0.05, and low-risk MIPI, p<0.0001). Accordingly, both IGHV 3-21 and 4-34 patients showed better long-term outcomes, with longer PFS and OS if compared with all the other cases, both singularly and when grouped together (3 years PFS: 75% vs 53%, p<0.05; 3 years OS: 91% vs 76%, p<0.01, Figure 1B, C). Finally, these results were independent from IGHV mutational status (p=0.09) and lenalidomide maintenance (p=0.46). Image:Summary/Conclusion: This is the first study assessing the clinical impact of VDJ IGH repertoire in a large prospective trial in MCL. As expected, 76% of patients resulted UM, with 3-21 and 4-34 representing more than one third of all the IGHV rearrangements. Notably, in our series, no significant correlation between mutational status and outcome was recorded. On the other hand, patients carrying either IGHV 3-21 or 4-34 rearrangements were characterized by favorable clinical and biological known prognosticators and were associated with both PFS and OS improvement, if compared to other patients. Deeper translational insights are needed to clarify the biological (autoreactive?) basis of this observation.

Abstract 2778: Validation of a next generation sequencing clinical assay for detection of immunoglobulin heavy chain clonality and somatic hypermutation in chronic lymphocytic leukemia

Abstract Next-generation sequencing (NGS) provides a powerful high-throughput approach to identify and track B-cell immunoglobulin (IG) heavy chain clonality and to assess somatic hypermutation status in a massively parallel manner. The NGS-based clonality/SHM testing demonstrated superior performance over the conventional capillary electrophoresis (CE) methods in characterization of B-cell neoplasms. However, its broad utilization in clinical diagnostics requires extensive validation of assays as well as standardization of data interpretation. We present here an NGS assay designed and validated specifically for IG clonality/SHM characterization of B-cell malignancies. The assay was designed to simultaneously target the Leader, FR1, FR2, and FR3 regions of the IGH gene to identify clonal IGH VH-JH rearrangement as well as the IGK gene to identify clonal IGK VK-JK, VK-Kde, and INTR-Kde rearrangement. The validation was performed to assess assay accuracy, specificity, sensitivity, repeatability, and reproducibility, with both pre-characterized reference controls and prospectively collected clinic peripheral blood and bone marrow aspirate specimen included. Triplicates were included and testing was performed at different times and by different operators to assess the assay precision. Over 60 clinic peripheral blood and bone marrow aspirate specimens were collected, including patients with B-cell malignancies such as CLL, B-ALL, and DLBCL as well as heathy donors, processed within 7 days for DNA extraction, and assessed by the NGS assay for IGH clonality and SHM assessment. Assay sensitivity as low as 2.5% for baseline clonality and as low as 0.001% for tracking MRD were observed. Near-perfect assay specificity and precision were observed at these sensitivity levels. The validated NGS assay was further qualified by ERIC (the European Research Initiative on CLL) with a certificate granted to standardize the data interpretation of this assay for testing in chronic lymphocytic leukemia. In conclusion, a NGS IG clonality/SHM assay was analytically and clinically validated under medical oversight, with a demonstrated rigor of the test by its high sensitivity/specificity and robust reproducibility. The clinical diagnostic testing results with this assay is interpreted in accordance with ERIC standards for reliable clinical reporting. Citation Format: Xin-Xing Tan, Tricia Peters, Meredith Berry, Kristyn Jeter, Brigitte Lovell, Victor Venegas, Yanglong Mou, Brad Thomas, Vincent Funari, Josette William. Validation of a next generation sequencing clinical assay for detection of immunoglobulin heavy chain clonality and somatic hypermutation in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2778.

Diagnostic Accuracy and Prognostic Relevance of Immunoglobulin Heavy Chain Rearrangement and 18F-FDG-PET/CT Compared With Unilateral Bone Marrow Trephination for Detecting Bone Marrow Involvement in Patients With Diffuse Large B-Cell Lymphoma.

BackgroundIn diffuse large B-cell lymphoma (DLBCL), bone marrow involvement (BMI) has an important clinical implication as a component of staging and International Prognostic Index. This study aimed to determine whether molecular analysis of immunoglobulin heavy chain (IgH) genes and positron emission tomography-computed tomography (PET/CT) could overcome the limitation of defining morphologic BMI by trephination biopsy and could increase the diagnostic accuracy or prognostic prediction.MethodsA total of 94 de novo patients with DLBCL underwent PET/CT, polymerase chain reaction (PCR) test for detection of IgH gene rearrangement, and unilateral bone marrow (BM) trephination at diagnosis.ResultsA total of 9 patients (9.6%) were confirmed to present morphologic BMI (mBMI) based on trephination biopsy. On the other hand, 21 patients (22.3%) were confirmed to have IgH clonality (IgH BMI), while 16 (17.0%) were classified with BMI based on the assessment of PET/CT (PET BMI). Each IgH rearrangement PCR and PET/CT showed the high negative predictive value of detecting the BMI. However, the combined assessment of IgH rearrangement and PET/CT could increase the diagnostic accuracy and specificity with 87.2% and 97.0%, respectively. The survival outcome of patients with double positive PET BMI and IgH BMI was significantly worse than that with either single positive PET BMI or IgH BMI, and even less than patients with neither PET BMI nor IgH BMI (3-year PFS: 50.0% vs. 75.4% vs. 97.9%, P = 0.007, 3-year OS: 50.0% vs. 75.6% vs. 80.1%, P = 0.035, respectively).ConclusionThis study suggests that the combined evaluation of PET/CT and IgH rearrangement could give additional information for predicting therapeutic outcomes in patients with negative morphologic BMI as an important part of the prognosis.

Impact of Maintenance Arm on Prognostic Value of MRD after Induction Treatment in MCL R2 Elderly Trial, a Mantle Cell Lymphoma Network Study

Impact of Maintenance Arm on Prognostic Value of MRD after Induction Treatment in MCL R2 Elderly Trial, a Mantle Cell Lymphoma Network Study

Targeted Locus Amplification (TLA): A Novel Next Generation Sequencing (NGS) Technology to Detect New Molecular Markers and Monitoring Minimal Residual Disease (MRD) in Mantle Cell and Follicular Lymphoma

Background: Minimal residual disease (MRD) monitoring by PCR methods is a strong and standardized predictor of clinical outcome in mantle cell (MCL) and follicular (FL) lymphoma [Pott C., Blood 2010; Ladetto M, Blood 2013]. However, some technical limitations hamper its feasibility in daily clinical routine. First of all, current techniques allow the identification of a molecular marker only in 75-80% of MCL and 55-60% of FL patients. All the other “no marker” cases cannot be studied for MRD purposes. Next generation sequencing (NGS) tools might overcome these limitations. Among them, the recently developed targeted locus amplification (TLA) technology [de Vree P.J., Nat Biotechnol 2014] that selectively amplifies and sequences entire genes on the basis of the crosslinking of physically proximal DNA loci, was able to identify novel candidate oncogenes and gene fusions in acute lymphoblastic leukemia. [Kuiper R.P.; Vroegindeweij EM, 57th ASH Communication, Blood 2015].The aim of this study was to test the TLA approach in MCL and FL diagnostic samples, with at least one translocation confirmed by FISH and a failure in molecular marker identification, with the aim to increase the rate of success in marker screening for MRD purposes. Moreover, the performances of the newly TLA molecular markers were compared to the standardized quantitative PCR (qPCR) approach for MRD monitoring.Methods: Genomic DNA was extracted from bone marrow (BM) and peripheral blood (PB) samples of MCL and FL patients, enrolled in prospective clinical trials of the Fondazione Italiana Linfomi (FIL). All patients provided written informed consent for the research use of the biological samples. Libraries for TLA (Cergentis, Utrecht) were prepared using only one couple of antisense primers targeting the immunoglobulin heavy chain (IGH) enhancer locus, as previously described [Hottentot QP, Methods Mol Biol. 2017], and sequenced on MiSeq platform (Illumina, San Diego). MRD monitoring was carried out by qPCR designing allele specific oligonucleotides (ASO) and consensus probes based on the new breakpoint sequences obtained from TLA NGS. Finally, the efficiency of TLA novel markers to monitor MRD was compared to MRD data obtained from clonal IGH rearrangements detected and tracked by standardized ASO qPCR. [Van der Velden VHJ, Leukemia 2007].Results: TLA was firstly tested on 21 t(11;14)-positive, BCL-1/IGH major translocation cluster (MTC) negative MCL baseline samples (8 BM and 13 PB): in 86% of cases (18/21) a novel BCL-1/IGH breakpoint was identified by NGS. In these cases, TLA described new rearrangements between “no-MTC” BCL1 loci and JH1, JH4, JH5 or JH6 regions on chromosome 14. Therefore, additional 8 FL baseline BM samples, resulting t(14;18) FISH positive and BCL-2/IGH MBR/mcr/“minor rearrangements”-negative by PCR, were tested obtaining a 62% (5 out of 8 samples) success rate. Also in these cases, new and not yet described BCL2/IGH rearrangements were found. Additionally, to assess the specificity of the technology, the MBR-positive control sample (DOHH2 cell line) was tested and correctly sequenced, as well. Interestingly, among the new TLA positive FL cases, in one sample, the NGS technology highlighted the breakpoint on the DH region of chromosome 14, describing an unusual t(14;18) translocation. 5/29 cases (17%) in which TLA was not able to identify a new marker were characterized by a low tumor infiltration, mainly <5% by flow cytometry (median 4.4%, range 0.03-8%). Secondly, a validation of the NGS sequences was performed by ASO primers designed on the new TLA breakpoints and MRD was monitored by ASO qPCR. Overall, in 14 MCL cases where also a classic IGH-based marker was available, the MRD results were superimposable between the two markers (Fig1A), with good and overlapping performances in terms of overall correlation (r2=0,86; Fig 1B). In FL samples, ASO qPCR confirmed the high tumor burden of the analyzed tissues (Fig 1C). Finally, an extension of the analyzed samples series and MRD validation is currently ongoing among additional FL patients.Conclusion: The TLA NGS technology allowed to identify a translocation-based novel molecular marker in FISH positive t(11;14) and t(14;18) MCL and FL baseline samples, where the classical Sanger sequencing failed a marker identification. These new breakpoints can reliably be used for the design of ASO qPCR primers for MRD detection in previously not traceable patients [Display omitted] DisclosuresKlous:Cergentis BV: Employment. Yilmaz:Cergentis BV: Employment. Vitolo:Gilead: Honoraria; Mundipharma: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria. Luminari:PFIZER: Speakers Bureau; GILEAD: Speakers Bureau; TEVA: Membership on an entity's Board of Directors or advisory committees; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees; ROCHE: Membership on an entity's Board of Directors or advisory committees; TAKEDA: Membership on an entity's Board of Directors or advisory committees. Federico:takeda: Honoraria, Research Funding. Splinter:Cergentis BV: Employment.

Immunoglobulin and T Cell Receptor β Chain Gene Rearrangement Analysis of Ocular Adnexal Lymphoid Neoplasms: Clinical and Biologic Implications

We investigated the organization of the immunoglobulin heavy chain (IgH) and the T cell receptor beta chain (T beta) gene loci in 20 ocular adnexal and four extraocular lymphoid neoplasms obtained from 18 patients presenting with an ocular adnexal lymphoid neoplasm. Fifteen ocular adnexal and four extraocular lymphoid neoplasms occurring in 13 patients were classified by morphological examination and immunophenotypic analysis as monoclonal B cell lymphomas. Each one of these 19 lymphoid neoplasms exhibited clonal IgH gene rearrangements upon hybridization of EcoRI- or HindIII-digested DNA to a heavy-chain joining region (JH)-specific DNA probe. The bilateral ocular adnexal monoclonal B cell neoplasms occurring simultaneously in two individuals exhibited identical clonal IgH gene rearrangements, which indicated their derivation from an identical B cell clone. The ocular adnexal and the extraocular monoclonal B cell neoplasms occurring in two of three patients also exhibited identical clonal IgH gene rearrangements, which suggested that they too were derived from an identical B cell clone. Five ocular adnexal lymphoid neoplasms were classified by morphological examination and immunophenotypic analysis as benign, polyclonal pseudolymphomas. Three of these five ocular adnexal lymphoid neoplasms exhibited clonal IgH gene rearrangements, which suggested the presence of monoclonal B cell populations that escaped detection by morphological and immunophenotypic examination. None of the 24 pathological samples exhibited clonal T beta gene rearrangements upon hybridization of EcoRI- or BamHI-digested DNA to a T beta gene DNA probe. The results of these studies demonstrate the value of Southern blot hybridization analysis for clonal IgH and T beta gene rearrangements in the diagnosis, classification, and investigation of extranodal lymphoid neoplasms originating and/or presenting in the ocular adnexa.

Abstract 171: Genomic and phenotypic analysis of post-transplant plasmablastic lymphomas

Abstract Introduction: Plasmablastic lymphoma (PBL), a rare and aggressive type of B-cell non-Hodgkin lymphoma, typically occurs in immunocompromised individuals, including a subset of patients who have undergone solid-organ transplantation. The pathogenesis of these lymphomas is not well understood and the underlying genetic alterations have not been characterized. In this study, we evaluated the genomic and pathologic features of PT-PBL to gain insights into disease biology. Methods: We searched our departmental database to identify PT-PBL diagnosed over the last 19 years. The pathologic features of each case and any prior lymphoproliferations were reviewed. A panel of 465 cancer-associated genes was sequenced using DNA extracted from each specimen and corresponding non-tumor tissue. Chromosomal aberrations involving CMYC and/or TP53 were assessed by fluorescence in situ hybridization (FISH). PCR analysis was performed to detect clonal immunoglobulin heavy chain (IGH) gene rearrangements. Results: Eighteen PT-lymphoproliferations from 11 solid organ allograft recipients (3 lung, 4 cardiac, 3 renal, and 1 combined liver and renal) with PT-PBL were identified. The median time to PBL development after transplant was 9.6 years (range 0.5 - 11.9 years). All PT-PBL occurred at extranodal sites and 4/11 (36%) were EBV+. Eight of 11 patients (73%) developed de novo PT-PBL, while in 3 cases, the PT-PBL was preceded by a clonally-related plasmacytoma-like lesion, a monomorphic PTLD, and a polymorphic PTLD. All cases showed plasmablastic morphology, with 7/11 (64%) showing variable degrees of plasmacytic differentiation. Nine of 11 (82%) PT-PBL expressed CD138. IgG, IgA or IgM was expressed by 4, 2, and 2 cases. CD56 or CD10 expression was noted in 2 and 5 cases, respectively. CMYC rearrangements were detected in 5/11 (45%) cases, 3 at the time of diagnosis and 2 upon disease progression. Next-generation sequencing revealed mutations in chromatin modifying genes (e.g. MLL2, MLL3, TET2) in 8/11 (73%) cases, RAS/MAP kinase pathway (e.g. KRAS, NRAS, HRAS) in 7/11 (64%) cases, JAK/STAT pathway (STAT3, STAT6, JAK3, SOCS1) in 4/11 (36%) cases, and the NOTCH pathway (e.g. NOTCH1, NOTCH3, SPEN) in 4/11 (36%) cases. DNA damage repair defects were seen in 8/11 (73%) cases, with 2/11 harboring mutations in DNA mismatch repair genes and showing microsatellite instability. Five of 11 (45%) PT-PBL had TP53 alterations; TP53 mutations at diagnosis (n=3), including 1 also exhibiting monosomy 17 (biallelic inactivation), an additional case with monosomy 17 at diagnosis, and one with TP53 deletion detected at relapse. Conclusions: Our results demonstrate different pathways of PT-PBL pathogenesis as well as immunophenotypic and genomic heterogeneity of these neoplasms. Furthermore, the observation of recurrent genetic alterations in PT-PBL that overlap with those described in diffuse large B-cell lymphomas and plasma cell neoplasms has implications for the design of effective therapies for these aggressive malignancies. Citation Format: Rebecca J. Leeman-Neill, Craig Soderquist, Murty Vundavalli, David Park, Susan Hsiao, Mahesh Mansukhani, Bachir Alobeid, Govind Bhagat. Genomic and phenotypic analysis of post-transplant plasmablastic lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 171.

Evaluation of Somatic Hypermutation Status in Chronic Lymphocytic Leukemia (CLL) in the Era of Next Generation Sequencing.

Somatic hypermutation (SHM) status provides an important prognostic indicator for chronic lymphocytic leukemia (CLL), a very common type of mature B-cell leukemia. Owing to the adverse prognosis associated with an unmutated immunoglobulin heavy chain variable (IGHV) status, SHM testing is performed as a standard of care in CLL. Conventionally, SHM testing has been performed using labor intensive and primarily analog Sanger sequencing method following PCR amplification of the clonal immunoglobulin heavy chain gene rearrangements in CLL cells. In comparison, recent availability of next generation sequencing (NGS) allows more versatile detection and direct identification of clonal immunoglobulin gene rearrangements in neoplastic B-cell populations. The ability to identify specific clonal IGHV signature(s) in both baseline (diagnostic) and post-treatment settings enables unique clinical applications of NGS such as determination of SHM status, minimal residual disease (MRD) monitoring, clonal heterogeneity and B cell receptor IG stereotypy. We provide a review of current practices and recommendations for SHM determination using NGS including examples of difficult cases.

Atypical Lymphoid Proliferations and Clonality in Helicobacter-associated Inflammatory Infiltrates in Children.

Helicobacter infection is considered the major predisposing factor for gastric mucosa-associated lymphoid tissue (MALT) lymphoma with initial infection likely occurring in childhood. Primary gastric MALT lymphoma most commonly occurs in patients older than 50 years which is attributed to the lengthy chronic infection time required before the development of MALT lymphoma. Our study analyzes the histologic features and presence of immunoglobulin heavy chain (IGH) clonality in Helicobacter-associated chronic gastritis (62 cases) and Helicobacter-negative chronic gastritis (17 cases) biopsies within the pediatric population, diagnosed between 1996 and 2018. Helicobacter-associated gastritis was more likely to show active inflammation (P=0.01), with no significant difference in number of germinal centers or the strength, linear property, or depth of the inflammatory infiltrate. In total, 47% (29/62) of the Helicobacter-associated cases had at least 1 lymphoepithelial lesion, equivocal or definitive (a modified Wotherspoon score of 3 to 5), compared with 24% (4/17) of the Helicobacter-negative cases (P=0.5). All cases with lymphoepithelial lesions were assessed for IGH clonality, showing the presence of monoclonality in 27% (8/30) of evaluable cases. None of our patients were diagnosed with gastric lymphoma within available follow-up data. Although 4% of our cases could be considered MALT lymphoma in an adult patient based on prominent lymphoepithelial lesions and IGH monoclonality, caution is advised when diagnosing lymphoma in the pediatric population given the good prognosis of Helicobacter-associated gastritis in this age group. It is unclear if these monoclonal lymphoid proliferations require close follow-up.

3142 Atypical Presentation of an Upper Gastrointestinal (GI) Bleed Manifesting as Extranodal Involvement of Diffuse Large B-Cell Lymphoma (DLBCL) Diagnosed in a Community Setting

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is the most common type of Non-Hodgkin's Lymphoma (NHL), with 5.4 new cases diagnosed per 100,000 persons as of 2016. There is a male predominance, with an average age of 64.2. One extranodal site involves the GI tract, predominantly the stomach. Commonly seen are mucosa associated lymphoid tissue lymphomas and DLBCL. The majority of cases present at stage III/IV, with 40% presenting as local involvement. Here we present an unusual case of extranodal DLBCL in a patient presenting with dysphagia and an upper GI bleed. CASE DESCRIPTION/METHODS: An 85 year-old female presented for evaluation of dysphonia, ongoing for several months. The patient complained of bilateral submandibular adenopathy, dysphagia, trouble breathing, and a 15 pound weight loss over two weeks. Computed tomography of the neck revealed a nasopharyngeal mass in the left (L) nasopharynx extending into the nasal cavity, right (R) nasopharynx, and eroding into the skull. Additionally, bilateral necrotic cervical lymph nodes and L supraclavicular adenopathy were also seen. Histologic sample was consistent with DLBCL, non-germinal center type. Fluorescence in-situ hybridization was negative for MYC rearrangement, t(8:14), t(4:18), and Epstein Barr Virus, but indeterminate for BCL6 rearrangement. She was staged IIA and underwent radiation therapy without chemotherapy. During treatment, the patient was admitted for evaluation of an upper GI bleed associated with dysphagia, fatigue, and progressive weakness. She underwent an esophagogastroduodenoscopy with findings showing Los Angeles class A esophagitis, large, inflammatory nodular tissue within the lesser curvature of the stomach with associated linear ulcerations, multiple punctate ulcers throughout the gastric body and antrum, duodenitis, and a small, clean-based ulcer in the duodenal sweep. Histology of the gastric mass revealed an atypical clonal lymphoid infiltrate involving the gastric mucosa, suggestive of DLBCL, with clonal immunoglobulin heavy chain gene rearrangement. DISCUSSION: This case represents an atypical presentation of an upper GI bleed that is the result of extranodal involvement of DLBCL. An atypical presentation like this should have lymphoma in the differential for those with a history. Treatment is dependent upon the extent of disease, usually starting with the regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone therapy. If left untreated, this disease can progress rapidly and prove fatal.

Clinical Utilization of Next-Generation Sequencing-Based Clonality Assays for Immunoglobulin Heavy Chain in Evaluating Suspected B-Cell Proliferation

Abstract Objectives B-cell clonality assays of the immunoglobulin heavy chain (IGH) and light chain (IGK) genes have been used widely in the workup of B-cell neoplasms. PCR is the current gold standard test for clonality. Recently, NGS has been suggested to both improve sensitivity and identify the specific V-(D-)J sequence required to track clones. Here we report our experience with using NGS for complementing our current procedures. Methods Our validated NGS test is performed by amplifying patients’ DNA using IGH-FR1 assay panel. Pooled amplicons get sequenced. The sequence data then get analyzed using in-house bioinformatics software, which sorts sequences and generates frequency distributions. Each sequence is ranked by its frequency. A clonal population is designated if the top sequence read is at least 1% of the total reads. For the standard PCR gene rearrangement test, we use a BIOMED-2 PCR-based B-cell assay. Results We retrospectively searched our database for all cases in which IGH and IGK clonality was tested by either standard PCR or NGS from 8/2016 to 7/2017. A total of 253 cases were tested by PCR while 21 cases were tested by NGS, which showed higher success rates. Of the 253 cases tested by conventional PCR, 39% (n = 98) were clonal (either IGH, IGK, or both), 34% (n = 86) were polyclonal, and the rest 28% (n = 69) showed a weak inconclusive signal or failed. Of the 21 cases analyzed by NGS, 71% (n = 15) were clonal, 24% (n = 5) were polyclonal, and just 5% (n = 1) failed. Conclusion Our experience indicates that NGS for IGH clonality, even more expensive, is an effective and more sensitive tool that produces higher informational content and yields higher success rates when compared to conventional PCR assay. NGS captures multidimensional data and provides a more precise determination of the clonal relationship between different lymphoid proliferations, which also is essential for the detection and measurement of minimal residual disease.

Prognostic markers for immunodeficiency-associated primary central nervous system lymphoma

Immunodeficiency is a major risk factor for primary central nervous system lymphoma (PCNSL), but data on the disease in immunocompromised hosts are scarce. We aimed to define clinical and imaging features and determine prognostic factors for immunodeficiency-associated PCNSL. All PCNSL cases seen at Yale-New Haven Hospital between 2002 and 2017 were retrospectively screened for immunodeficiency. For patients with immunosuppression, biopsies were evaluated and clinical data were collected. Predictors of survival were identified using Kaplan-Meier survival analysis and log-rank test. p values < 0.05 were considered significant. 23 patients with immunodeficiencies were identified: eleven on immunosuppressants after solid organ transplantation, seven with human immunodeficiency virus infection, and five on immunosuppressive treatment due to various autoimmune disorders. PCNSL cases were largely Epstein-Barr-Virus positive (78%), histologically classified as diffuse large B cell lymphomas (87%), and showed peripheral contrast enhancement (81%) and corresponding heterogeneous diffusion-weighted imaging patterns (DWI) on magnetic resonance imaging (MRI) (71%). Median overall survival was 31months. Age > 60years at diagnosis (p < 0.01), peripheral enhancement of the mass on MRI (p = 0.04), heterogeneous DWI patterns (p = 0.04), and clonal immunoglobulin heavy chain gene rearrangement (IgHR) (p = 0.03) were found to be negative prognostic markers. Immunodeficiency-associated PCNSL presents with similar clinical, pathological and imaging features. Age > 60years, clonal IgHR, heterogeneous DWI pattern and peripheral enhancement on MRI may serve as predictors of less favorable outcome.